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2.
Nat Commun ; 12(1): 34, 2021 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-33397947

RESUMO

Colloidal gold nanoparticles (GNPs) serve as promising contrast agents in photoacoustic (PA) imaging, yet their utility is limited due to their absorption peak in the visible window overlapping with that of hemoglobin. To overcome such limitation, this report describes an ultrapure chain-like gold nanoparticle (CGNP) clusters with a redshift peak wavelength at 650 nm. The synthesized CGNP show an excellent biocompatibility and photostability. These nanoparticles are conjugated with arginine-glycine-aspartic acid (RGD) peptides (CGNP clusters-RGD) and validated in 12 living rabbits to perform multimodal photoacoustic microscopy (PAM) and optical coherence tomography (OCT) for visualization of newly developed blood vessels in the sub-retinal pigment epithelium (RPE) space of the retina, named choroidal neovascularization (CNV). The PAM system can achieve a 3D PAM image via a raster scan of 256 × 256 pixels within a time duration of 65 s. Intravenous injection of CGNP clusters-RGD bound to CNV and resulted in up to a 17-fold increase in PAM signal and 176% increase in OCT signal. Histology indicates that CGNP clusters could disassemble, which may facilitate its clearance from the body.


Assuntos
Ouro/química , Nanopartículas Metálicas/química , Microscopia , Imagem Molecular , Técnicas Fotoacústicas , Tomografia de Coerência Óptica , Animais , Neovascularização de Coroide/diagnóstico por imagem , Neovascularização de Coroide/patologia , Meios de Contraste/química , Feminino , Testes de Função Renal , Testes de Função Hepática , Masculino , Nanopartículas Metálicas/ultraestrutura , Camundongos , Oligopeptídeos/química , Coelhos , Distribuição Tecidual
3.
Nat Commun ; 12(1): 162, 2021 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-33420085

RESUMO

Guanine rich regions of oligonucleotides fold into quadruple-stranded structures called G-quadruplexes (G4s). Increasing evidence suggests that these G4 structures form in vivo and play a crucial role in cellular processes. However, their direct observation in live cells remains a challenge. Here we demonstrate that a fluorescent probe (DAOTA-M2) in conjunction with fluorescence lifetime imaging microscopy (FLIM) can identify G4s within nuclei of live and fixed cells. We present a FLIM-based cellular assay to study the interaction of non-fluorescent small molecules with G4s and apply it to a wide range of drug candidates. We also demonstrate that DAOTA-M2 can be used to study G4 stability in live cells. Reduction of FancJ and RTEL1 expression in mammalian cells increases the DAOTA-M2 lifetime and therefore suggests an increased number of G4s in these cells, implying that FancJ and RTEL1 play a role in resolving G4 structures in cellulo.


Assuntos
DNA/metabolismo , Quadruplex G , Microscopia Intravital/métodos , Imagem Molecular/métodos , Animais , Linhagem Celular Tumoral , DNA/química , DNA Helicases/genética , DNA Helicases/metabolismo , Proteínas de Grupos de Complementação da Anemia de Fanconi/genética , Proteínas de Grupos de Complementação da Anemia de Fanconi/metabolismo , Fibroblastos , Corantes Fluorescentes/química , Técnicas de Silenciamento de Genes , Humanos , Indóis/química , Camundongos , Microscopia de Fluorescência/métodos , RNA Helicases/genética , RNA Helicases/metabolismo
5.
Nat Commun ; 12(1): 717, 2021 01 29.
Artigo em Inglês | MEDLINE | ID: mdl-33514717

RESUMO

The Neisseria meningitidis protein FrpC contains a self-processing module (SPM) undergoing autoproteolysis via an aspartic anhydride. Herein, we establish NeissLock, using a binding protein genetically fused to SPM. Upon calcium triggering of SPM, the anhydride at the C-terminus of the binding protein allows nucleophilic attack by its target protein, ligating the complex. We establish a computational tool to search the Protein Data Bank, assessing proximity of amines to C-termini. We optimize NeissLock using the Ornithine Decarboxylase/Antizyme complex. Various sites on the target (α-amine or ε-amines) react with the anhydride, but reaction is blocked if the partner does not dock. Ligation is efficient at pH 7.0, with half-time less than 2 min. We arm Transforming Growth Factor-α with SPM, enabling specific covalent coupling to Epidermal Growth Factor Receptor at the cell-surface. NeissLock harnesses distinctive protein chemistry for high-yield covalent targeting of endogenous proteins, advancing the possibilities for molecular engineering.


Assuntos
Proteínas de Bactérias/genética , Proteínas de Membrana/genética , Sondas Moleculares/metabolismo , Engenharia de Proteínas/métodos , Proteínas Recombinantes de Fusão/metabolismo , Coloração e Rotulagem/métodos , Anidridos/metabolismo , Animais , Imagem Molecular/métodos , Sondas Moleculares/química , Sondas Moleculares/genética , Proteólise , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética
6.
Nat Commun ; 12(1): 716, 2021 01 29.
Artigo em Inglês | MEDLINE | ID: mdl-33514737

RESUMO

For over two decades photoacoustic imaging has been tested clinically, but successful human trials have been limited. To enable quantitative clinical spectroscopy, the fundamental issues of wavelength-dependent fluence variations and inter-wavelength motion must be overcome. Here we propose a real-time, spectroscopic photoacoustic/ultrasound (PAUS) imaging approach using a compact, 1-kHz rate wavelength-tunable laser. Instead of illuminating tissue over a large area, the fiber-optic delivery system surrounding an US array sequentially scans a narrow laser beam, with partial PA image reconstruction for each laser pulse. The final image is then formed by coherently summing partial images. This scheme enables (i) automatic compensation for wavelength-dependent fluence variations in spectroscopic PA imaging and (ii) motion correction of spectroscopic PA frames using US speckle tracking in real-time systems. The 50-Hz video rate PAUS system is demonstrated in vivo using a murine model of labelled drug delivery.


Assuntos
Sistemas Computacionais , Imagem Molecular/métodos , Técnicas Fotoacústicas/métodos , Análise Espectral/métodos , Animais , Desenho de Equipamento , Feminino , Processamento de Imagem Assistida por Computador , Lasers , Camundongos , Camundongos Nus , Modelos Animais , Imagem Molecular/instrumentação , Movimento (Física) , Fibras Ópticas , Imagens de Fantasmas , Técnicas Fotoacústicas/instrumentação , Análise Espectral/instrumentação , Ultrassonografia/instrumentação , Ultrassonografia/métodos
7.
Food Chem ; 334: 127586, 2021 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-32707364

RESUMO

It is unknown whether intestinal absorption of acylated anthocyanins occurs in their intact or metabolized form. In this study, with the aid of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) imaging, intestinal absorption of acylated anthocyanins was visually investigated. Anthocyanin extracts from purple carrots were orally administered to Sprague-Dawley rats. Acylated cyanidins were absorbed into portal and circulating blood systems in their intact form, and aglycon; cyanidin 3-O-(6-O-feruloyl-ß-d-glucopyranosyl)-(1 â†’ 6)-[ß-d-xylopyranosyl-(1 â†’ 2)]-ß-d-galactopyranoside (Cy3XFGG), and showed a high absorption of 39.3 ± 0.1 pmol/mL-plasma at 60 min after administration. MALDI-MS imaging analysis of the rat jejunum membranes showed that an organic anion transporting polypeptide (OATP) transporter was involved in Cy3XFGG transport, while deacylated anthocyanins were incorporated through both the glucose transporter 2 and OATP routes. In conclusion, acylated anthocyanin, Cy3XFGG, can be absorbed in its intact form through intestinal OATP.


Assuntos
Antocianinas/análise , Antocianinas/farmacocinética , Imagem Molecular/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Acilação , Administração Oral , Animais , Antocianinas/administração & dosagem , Cor , Daucus carota/química , Absorção Intestinal/efeitos dos fármacos , Jejuno/efeitos dos fármacos , Jejuno/metabolismo , Masculino , Transportadores de Ânions Orgânicos/metabolismo , Extratos Vegetais/administração & dosagem , Extratos Vegetais/farmacocinética , Ratos Sprague-Dawley
8.
Anal Chem ; 93(4): 2045-2052, 2021 02 02.
Artigo em Inglês | MEDLINE | ID: mdl-33326221

RESUMO

Apoptosis plays an essential role in a multicellular organism's lifecycle. Developing technologies for selectively monitoring apoptotic processes can be useful not only in the evaluation of disease progression, but also in the assessment of their therapeutic intervention. However, quantitative imaging of cell apoptosis is still a challenge. In this work, we reported a cell-permeable peptide probe with a ratiometric fluorescence response specifically toward caspase-3, a key enzyme for the execution of apoptosis. This probe Ac-Tat-DEVD-CV consisted of a caspase-3 recognition sequence Asp-Glu-Val-Asp (DEVD), a cell-penetrating peptide Tat (RKKRRORRR), and a long wavelength fluorophore, cresyl violet (CV). Upon selective hydrolyzation by caspase-3, the probe released CV and displayed a ratiometric change in fluorescence. Facilitated by the cell-penetrating peptide, this probe can easily internalize into cells. The ratiometric response property bestowed the probe with advantages in the real-time quantification of caspase-3 activity, thus estimating the apoptotic stages in living cells. This method could offer opportunities to evaluate apoptosis-related disease progression and therapeutic monitoring.


Assuntos
Caspase 3/química , Caspase 3/metabolismo , Corantes Fluorescentes/química , Imagem Molecular/métodos , Imagem Óptica/métodos , Peptídeos/química , Células HeLa , Humanos
9.
Chimia (Aarau) ; 74(12): 953-959, 2020 Dec 23.
Artigo em Inglês | MEDLINE | ID: mdl-33357288

RESUMO

Due to its long half-life of 2.111×105 y, technetium, i.e. 99Tc, offers the excellent opportunity of combining fundamental and ' classical ' organometallic or coordination chemistry with all methodologies of radiochemistry. Technetium chemistry is inspired by the applications of its short-lived metastable isomer 99mTc in molecular imaging and radiopharmacy. We present in this article examples about these contexts and the impact of purely basic oriented research on practical applications. This review shows how the chemistry of this element in the middle of the periodic system inspires the chemistry of neighboring elements such as rhenium. Reasons are given for the frequent observation that the chemistries of 99Tc and 99mTc are often not identical, i.e. compounds accessible for 99mTc, under certain conditions, are not accessible for 99Tc. The article emphasizes the importance of macroscopic technetium chemistry not only for research but also for advanced education in the general fields of radiochemistry.


Assuntos
Imagem Molecular , Rênio , Radioquímica , Compostos Radiofarmacêuticos , Tecnécio
10.
PLoS One ; 15(12): e0243742, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33347445

RESUMO

Recently, human asparagine synthetase has been found to be associated with the mitotic spindle. However, this event cannot be seen in yeast because yeast takes a different cell division process via closed mitosis (there is no nuclear envelope breakdown to allow the association between any cytosolic enzyme and mitotic spindle). To find out if yeast asparagine synthetase can also (but hiddenly) have this feature, the coding sequences of green fluorescent protein (GFP) and nuclear localization signal (NLS) were introduced downstream of ASN1 and ASN2, encoding asparagine synthetases Asn1p and Asn2p, respectively, in the yeast genome having mCherrry coding sequence downstream of TUB1 encoding alpha-tubulin, a building block of the mitotic spindle. The genomically engineered yeast strains showed co-localization of Asn1p-GFP-NLS (or Asn2p-GFP-NLS) and Tub1p-mCherry in dividing nuclei. In addition, an activity-disrupted mutation was introduced to ASN1 (or ASN2). The yeast mutants still exhibited co-localization between defective asparagine synthetase and mitotic spindle, indicating that the biochemical activity of asparagine synthetase is not required for its association with the mitotic spindle. Furthermore, nocodazole treatment was used to depolymerize the mitotic spindle, resulting in lack of association between the enzyme and the mitotic spindle. Although yeast cell division undergoes closed mitosis, preventing the association of its asparagine synthetase with the mitotic spindle, however, by using yeast constructs with re-localized Asn1/2p have suggested the moonlighting role of asparagine synthetase in cell division of higher eukaryotes.


Assuntos
Aspartato-Amônia Ligase/metabolismo , Carbono-Nitrogênio Ligases com Glutamina como Doadora de N-Amida/metabolismo , Mitose/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiologia , Fuso Acromático/metabolismo , Aspartato-Amônia Ligase/genética , Carbono-Nitrogênio Ligases com Glutamina como Doadora de N-Amida/genética , Núcleo Celular/metabolismo , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Microscopia Intravital/métodos , Substâncias Luminescentes/química , Proteínas Luminescentes/química , Proteínas Luminescentes/genética , Microscopia de Fluorescência , Imagem Molecular/métodos , Proteínas de Saccharomyces cerevisiae/genética
11.
Science ; 370(6523)2020 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-33335032

RESUMO

Myelin plasticity is critical for neurological function, including learning and memory. However, it is unknown whether this plasticity reflects uniform changes across all neuronal subtypes, or whether myelin dynamics vary between neuronal classes to enable fine-tuning of adaptive circuit responses. We performed in vivo two-photon imaging of myelin sheaths along single axons of excitatory callosal neurons and inhibitory parvalbumin-expressing interneurons in adult mouse visual cortex. We found that both neuron types show homeostatic myelin remodeling under normal vision. However, monocular deprivation results in adaptive myelin remodeling only in parvalbumin-expressing interneurons. An initial increase in elongation of myelin segments is followed by contraction of a separate cohort of segments. This data indicates that distinct classes of neurons individualize remodeling of their myelination profiles to diversify circuit tuning in response to sensory experience.


Assuntos
Bainha de Mielina/metabolismo , Neocórtex/metabolismo , Neurônios/metabolismo , Córtex Visual/metabolismo , Animais , Corpo Caloso/citologia , Corpo Caloso/metabolismo , Feminino , Neurônios GABAérgicos/metabolismo , Interneurônios/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Imagem Molecular , Neocórtex/citologia , Plasticidade Neuronal , Neurônios/classificação , Parvalbuminas/metabolismo , Córtex Visual/citologia
12.
Yakugaku Zasshi ; 140(11): 1299-1303, 2020.
Artigo em Japonês | MEDLINE | ID: mdl-33132264

RESUMO

The author has developed several methodological approaches that use nanophotonic and microfluidic devices to accelerate pharmaceutical research and development. Here, the author describes two of these approaches and provides practical examples. The first is a nanophotonic approach to break the concentration limit of diffusing fluorophore-labeled molecules in single-molecule imaging. Although single-molecule imaging is highly useful in characterizing the kinetics of biomolecular interactions, it requires nanomolar concentrations of labeled molecules in solution. Zero-mode waveguides are nanophotonic structures that reduce the illumination volume by more than three orders of magnitude relative to conventional fluorescence microscopy, thereby allowing single-molecule investigations at micromolar to millimolar concentrations of fluorescent molecules i.e., under near-physiological conditions. The second approach is microfluidic microdroplet-based, allowing the discovery of novel biomolecules with the desired activities. Microfluidics allows the ultrarapid production of monodisperse microdroplets such as water-in-oil microdroplets. Each microdroplet serves as a nano/picoliter-volume test tube, which increases assay sensitivity by increasing the effective concentration of molecules and decreasing the time required to reach detection thresholds. I hope you find this review helpful in your research.


Assuntos
Corantes Fluorescentes , Técnicas Analíticas Microfluídicas/instrumentação , Microscopia de Fluorescência , Imagem Molecular/métodos , Nanotecnologia/instrumentação , Pesquisa Farmacêutica/instrumentação , Pesquisa Farmacêutica/métodos
13.
BMC Bioinformatics ; 21(1): 448, 2020 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-33036551

RESUMO

BACKGROUND: Multimodal imaging that combines mass spectrometry imaging (MSI) with Raman imaging is a rapidly developing multidisciplinary analytical method used by a growing number of research groups. Computational tools that can visualize and aid the analysis of datasets by both techniques are in demand. RESULTS: Raman2imzML was developed as an open-source converter that transforms Raman imaging data into imzML, a standardized common data format created and adopted by the mass spectrometry community. We successfully converted Raman datasets to imzML and visualized Raman images using open-source software designed for MSI applications. CONCLUSION: Raman2imzML enables both MSI and Raman images to be visualized using the same file format and the same software for a straightforward exploratory imaging analysis.


Assuntos
Processamento de Imagem Assistida por Computador/normas , Espectrometria de Massas , Imagem Molecular , Análise Espectral Raman , Padrões de Referência
14.
Nat Commun ; 11(1): 5107, 2020 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-33037199

RESUMO

Engineered light-dependent switches provide uniquely powerful opportunities to investigate and control cell regulatory mechanisms. Existing tools offer high spatiotemporal resolution, reversibility and repeatability. Cellular optogenetics applications remain limited with diffusible targets as the response of the actuator is difficult to independently validate. Blue light levels commonly needed for actuation can be cytotoxic, precluding long-term experiments. We describe a simple approach overcoming these obstacles. Resonance energy transfer can be used to constitutively or dynamically modulate actuation sensitivity. This simultaneously offers on-line monitoring of light-dependent switching and precise quantification of activation-relaxation properties in intact living cells. Applying this approach to different LOV2-based switches reveals that flanking sequences can lead to relaxation times up to 11-fold faster than anticipated. In situ-measured parameter values guide the design of target-inhibiting actuation trains with minimal blue-light exposure, and context-based optimisation can increase sensitivity and experimental throughput a further 10-fold without loss of temporal precision.


Assuntos
Imagem Molecular/métodos , Optogenética/métodos , Fototropinas/metabolismo , Animais , Transferência de Energia , Feminino , Transferência Ressonante de Energia de Fluorescência/métodos , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Hipocampo/citologia , Humanos , Luz , Sistema de Sinalização das MAP Quinases , Masculino , Neurônios , Fototropinas/análise , Fototropinas/genética , Ratos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo
15.
Radiol Clin North Am ; 58(6): 1135-1146, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-33040853

RESUMO

This article is a summary of the most up-to-date applications of radiopharmaceuticals to the diagnosis and therapy of benign and malignant diseases involving endocrine or neuroendocrine organs of the head and neck, focusing on radiotracers approved by the US Food and Drug Administration, such as I-123- and I-131-sodium iodide, F-18-fluorodeoxyglucose, Tc99m-sestamibi, as well as the more recently approved tracers Ga-68 DOTATATE and Lu-177 DOTATATE.


Assuntos
Neoplasias de Cabeça e Pescoço/diagnóstico por imagem , Imagem Molecular/métodos , Tumores Neuroendócrinos/diagnóstico por imagem , Tomografia por Emissão de Pósitrons/métodos , Compostos Radiofarmacêuticos/farmacologia , Feminino , Neoplasias de Cabeça e Pescoço/patologia , Neoplasias de Cabeça e Pescoço/terapia , Humanos , Radioisótopos do Iodo/farmacologia , Masculino , Tumores Neuroendócrinos/patologia , Tumores Neuroendócrinos/terapia , Compostos Organometálicos/farmacologia , Sensibilidade e Especificidade , Estados Unidos , United States Food and Drug Administration
16.
Neuron ; 108(1): 93-110, 2020 10 14.
Artigo em Inglês | MEDLINE | ID: mdl-33058769

RESUMO

Visualizing and perturbing neural activity on a brain-wide scale in model animals and humans is a major goal of neuroscience technology development. Established electrical and optical techniques typically break down at this scale due to inherent physical limitations. In contrast, ultrasound readily permeates the brain, and in some cases the skull, and interacts with tissue with a fundamental resolution on the order of 100 µm and 1 ms. This basic ability has motivated major efforts to harness ultrasound as a modality for large-scale brain imaging and modulation. These efforts have resulted in already-useful neuroscience tools, including high-resolution hemodynamic functional imaging, focused ultrasound neuromodulation, and local drug delivery. Furthermore, recent breakthroughs promise to connect ultrasound to neurons at the genetic level for biomolecular imaging and sonogenetic control. In this article, we review the state of the art and ongoing developments in ultrasonic neurotechnology, building from fundamental principles to current utility, open questions, and future potential.


Assuntos
Encéfalo/diagnóstico por imagem , Sistemas de Liberação de Medicamentos/métodos , Ecoencefalografia/métodos , Imagem Molecular/métodos , Ondas Ultrassônicas , Animais , Barreira Hematoencefálica/efeitos da radiação , Encéfalo/fisiologia , Encéfalo/efeitos da radiação , Neuroimagem Funcional , Hemodinâmica , Humanos , Proteínas , Terapia por Ultrassom , Ultrassonografia , Ultrassonografia Doppler Transcraniana/métodos
17.
J Vis Exp ; (163)2020 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-33016934

RESUMO

Periosteal skeletal stem cells (P-SSCs) are essential for lifelong bone maintenance and repair, making them an ideal focus for the development of therapies to enhance fracture healing.  Periosteal cells rapidly migrate to an injury to supply new chondrocytes and osteoblasts for fracture healing. Traditionally, the efficacy of a cytokine to induce cell migration has only been conducted in vitro by performing a transwell or scratch assay. With advancements in intravital microscopy using multiphoton excitation, it was recently discovered that 1) P-SSCs express the migratory gene CCR5 and 2) treatment with the CCR5 ligand known as CCL5 improves fracture healing and the migration of P-SSCs in response to CCL5. These results have been captured in real-time. Described here is a protocol to visualize P-SSC migration from the calvarial suture skeletal stem cell (SSC) niche towards an injury after treatment with CCL5. The protocol details the construction of a mouse restraint and imaging mount, surgical preparation of the mouse calvaria, induction of a calvaria defect, and acquisition of time-lapse imaging.


Assuntos
Movimento Celular/efeitos dos fármacos , Quimiocina CCL5/farmacologia , Imagem Molecular , Periósteo/citologia , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Animais , Camundongos , Fatores de Tempo
18.
J Vis Exp ; (162)2020 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-32865536

RESUMO

Neonates are at an increased risk of bacterial sepsis due to the unique immune profile they display in the first months of life. We have established a protocol for studying the pathogenesis of E. coli O1:K1:H7, a serotype responsible for high mortality rates in neonates. Our method utilizes intravital imaging of neonatal pups at different time points during the progression of infection. This imaging, paralleled by measurement of bacteria in the blood, inflammatory profiling, and tissue histopathology, signifies a rigorous approach to understanding infection dynamics during sepsis. In the current report, we model two infectious inoculums for comparison of bacterial burdens and severity of disease. We find that subscapular infection leads to disseminated infection by 10 h post-infection. By 24 h, infection of luminescent E. coli was abundant in the blood, lungs, and other peripheral tissues. Expression of inflammatory cytokines in the lungs is significant at 24 h, and this is followed by cellular infiltration and evidence of tissue damage that increases with infectious dose. Intravital imaging does have some limitations. This includes a luminescent signal threshold and some complications that can arise with neonates during anesthesia. Despite some limitations, we find that our infection model offers an insight for understanding longitudinal infection dynamics during neonatal murine sepsis, that has not been thoroughly examined to date. We expect this model can also be adapted to study other critical bacterial infections during early life.


Assuntos
Infecções por Escherichia coli/diagnóstico por imagem , Escherichia coli/fisiologia , Imagem Molecular , Sepse/diagnóstico por imagem , Animais , Animais Recém-Nascidos , Modelos Animais de Doenças , Camundongos
19.
Nucleic Acids Res ; 48(17): 9694-9709, 2020 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-32890402

RESUMO

DNA breaks recruit and activate PARP1/2, which deposit poly-ADP-ribose (PAR) to recruit XRCC1-Ligase3 and other repair factors to promote DNA repair. Clinical PARP inhibitors (PARPi) extend the lifetime of damage-induced PARP1/2 foci, referred to as 'trapping'. To understand the molecular nature of 'trapping' in cells, we employed quantitative live-cell imaging and fluorescence recovery after photo-bleaching. Unexpectedly, we found that PARP1 exchanges rapidly at DNA damage sites even in the presence of clinical PARPi, suggesting the persistent foci are not caused by physical stalling. Loss of Xrcc1, a major downstream effector of PAR, also caused persistent PARP1 foci without affecting PARP1 exchange. Thus, we propose that the persistent PARP1 foci are formed by different PARP1 molecules that are continuously recruited to and exchanging at DNA lesions due to attenuated XRCC1-LIG3 recruitment and delayed DNA repair. Moreover, mutation analyses of the NAD+ interacting residues of PARP1 showed that PARP1 can be physically trapped at DNA damage sites, and identified H862 as a potential regulator for PARP1 exchange. PARP1-H862D, but not PARylation-deficient PARP1-E988K, formed stable PARP1 foci upon activation. Together, these findings uncovered the nature of persistent PARP1 foci and identified NAD+ interacting residues involved in the PARP1 exchange.


Assuntos
Dano ao DNA , Reparo do DNA/efeitos dos fármacos , Poli(ADP-Ribose) Polimerase-1/genética , Poli(ADP-Ribose) Polimerase-1/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Sítios de Ligação , Domínio Catalítico , Linhagem Celular Tumoral , Reparo do DNA/fisiologia , Transferência Ressonante de Energia de Fluorescência , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Indazóis/farmacologia , Cinética , Imagem Molecular , NAD/metabolismo , Piperidinas/farmacologia , Poli(ADP-Ribose) Polimerases/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteína 1 Complementadora Cruzada de Reparo de Raio-X/genética , Proteína 1 Complementadora Cruzada de Reparo de Raio-X/metabolismo
20.
Int J Nanomedicine ; 15: 6721-6734, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32982229

RESUMO

Introduction: Oxaliplatin (L-OHP) is a well-known third-generation platinum anticancer drug with severe systemic- and neuro-toxicity. The main objective of the current research was to develop a targeted long-circulating thermosensitive smart-release liposome (LCTL) system for better therapeutic efficacy and less toxicity. Methods: The reverse-phase evaporation method (REV) was used to prepare L-OHP loaded LCTL (L-OHP/LCTL). The physical characteristics were evaluated including encapsulation efficiency (EE), size, zeta potential and stability. The release behavior, cytotoxicity and in vivo evaluation were also carried out. Results: EE of LCTL was around 25% with a uniform size distribution, and LCTL achieved almost complete release at 42°C while it was only 10% at 37°C. Moreover, the LCTL showed significantly higher cytotoxicity at 42°C than that at 37°C. The in vivo results indicated LCTL could target tumors and enhance retention for more than 24 h, thereby enhancing anti-tumor efficacy on 4T1-bearing mice. Discussion: These results indicated that LCTL not only possessed a prolonged circulation time but it also enhanced accumulation and achieved selective release at the tumor sites. Conclusively, LCTL could serve as a promising carrier for oxaliplatin delivery to treat solid tumors.


Assuntos
Antineoplásicos/administração & dosagem , Sistemas de Liberação de Medicamentos/métodos , Lipossomos/administração & dosagem , Lipossomos/química , Oxaliplatina/administração & dosagem , Animais , Antineoplásicos/farmacocinética , Linhagem Celular Tumoral , Estabilidade de Medicamentos , Humanos , Lipossomos/uso terapêutico , Masculino , Camundongos Endogâmicos BALB C , Imagem Molecular , Neoplasias/tratamento farmacológico , Oxaliplatina/farmacocinética , Tamanho da Partícula , Coelhos , Temperatura , Ensaios Antitumorais Modelo de Xenoenxerto
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